TLR4, the predominant signaling receptor for LPS, is critical for host defense against Gram negative bacterial infections. Two point mutations, D299G and T399I, in the ectodomain of TLR4 have been associated with a blunted response to inhaled LPS, increased incidence of Gram negative bacterial infection and sepsis. These results suggest that these mutations alter the capacity of TLR4 to signal, leading to impaired host immune response against bacterial pathogens. The molecular mechanisms by which TLR4 polymorphisms affect receptor-mediated signaling are currently unknown. The central hypothesis to be tested is that the single nucleotide polymorphisms alter TLR4 recognition of Gram negative bacteria and LPS, its interactions with co-receptors and recruitment and activation of adapters and kinases, resulting in impaired TLR4 signaling and deficient host immune response against pathogens. This hypothesis will be examined by pursuit of the following Specific Aims: (1) Define whether the TLR4 SNPs alter recognition of Gram negative bacteria and LPS, receptor signalosome assembly, and activation of kinases and transcription factors;(2) Determine the effect of the TLR4 SNPs on expression of inflammatory mediators and phagocytosis of Gram negative bacteria. Complementary approaches will be used to conduct our studies, including transfected cell lines, primary TLR4-/- mouse macrophages with "knocked-in" human wild-type or mutant TLR4s introduced by nucleofection, and human monocytes obtained from human subjects who carry wild-type or mutant TLR4 alleles. At the completion of these studies, we expect to identify molecular mechanisms by which TLR4 mutations affect sensing of Gram negative bacteria and LPS. This research will advance our general understanding of TLR4 signaling, and provide a rationale for the development of new therapeutic approaches to correct functions of components of the TLR signaling pathway compromised in individuals expressing mutant TLR4 variants. PUBLIC HEALTH RELEVANCE: Bacterial sepsis is a major threat to human health worldwide, with the annual incidence in the U.S.A. is ~750,000 patients, with more than ~210,000 (>28%) deaths. Mutations in TLR4, the main receptor for Gram negative bacteria and lipopolysaccharide, have been associated with many infectious diseases and sepsis. However, molecular mechanisms by which these mutations affect receptor signaling and macrophage reactions to microbes are unknown. Our proposed studies will determine how these TLR4 mutations change receptor functions at the molecular level. Our research will identify earliest disturbances in functions of receptor and signaling molecules that are caused by TLR4 mutations, and may aid to development of new pharmaceutical drugs to correct such deficiencies.